Nucleoside-based inhibitors of the essential HIV-1 enzyme reverse transcriptase (RT) prolong survival in patients with AIDS and delay disease progression in patients with earlier stages of HIV infection. Although an increasing number of HIV-1 isolates with reduced susceptibility to zidovudine (ZDV), dideoxyinosine (ddI), and dideoxycytidine (ddC) are being reported, the clinical significance of viral drug resistance is still unproven. Nevertheless, it is likely that the emergence of a pool of virus with reduced susceptibility to RT inhibitors is responsible, at least in part, for disease progression in patients treated with these drugs. Thus, testing drug susceptibility is likely to assume an increasingly important place in the design of clinical trials of antiretroviral therapy and in clinical practice. As in other infectious diseases, the use of combination chemotherapy may prevent the emergence of drug resistance and minimize toxicity. However, in vitro synergy studies reported to date have used tissue culture-adapted strains of HIV-1 that may have different biological properties as compared to clinical isolates. As participants in the ACTG Virology Resistance Working Group, we propose to develop and test a standardized protocol for testing drug-susceptibility of low-passage clinical isolates of HIV-1 from patients enrolled in ACTG trials of antiretroviral agents such as ZDV, ddI, ddC, and L-697,661. RT genes from drug-resistant isolates will be analyzed by selective PCR amplification or by cloning and sequencing to detect genotypic changes associated with drug resistance. The effect of in vitro selective pressure on the emergence of high-level drug resistance from populations of HIV-1 with intermediate drug susceptibility or mixed genotypes will be studied. Combinations of antiretroviral agents will be tested in vitro for synergy using low-passage HIV-1 isolates with reduced sensitivity to one or more drugs.